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      Dopamine Transporter Phosphorylation Site Threonine 53 Regulates Substrate Reuptake and Amphetamine-stimulated Efflux*

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          Abstract

          Background: DAT activity is regulated by protein kinases.

          Results: We identify Thr 53 as a DAT phosphorylation site in rat striatum by mass spectrometry and a phospho-specific antibody; Thr 53 mutation reduced dopamine influx and ablated transporter-mediated efflux.

          Conclusion: Phosphorylation of DAT Thr 53 is involved in transport activity.

          Significance: These results identify Thr 53 phosphorylation of DAT in vivo and elucidate associated functional properties.

          Abstract

          In the central nervous system, levels of extraneuronal dopamine are controlled primarily by the action of the dopamine transporter (DAT). Multiple signaling pathways regulate transport activity, substrate efflux, and other DAT functions through currently unknown mechanisms. DAT is phosphorylated by protein kinase C within a serine cluster at the distal end of the cytoplasmic N terminus, whereas recent work in model cells revealed proline-directed phosphorylation of rat DAT at membrane-proximal residue Thr 53. In this report, we use mass spectrometry and a newly developed phospho-specific antibody to positively identify DAT phosphorylation at Thr 53 in rodent striatal tissue and heterologous expression systems. Basal phosphorylation of Thr 53 occurred with a stoichiometry of ∼50% and was strongly increased by phorbol esters and protein phosphatase inhibitors, demonstrating modulation of the site by signaling pathways that impact DAT activity. Mutations of Thr 53 to prevent phosphorylation led to reduced dopamine transport V max and total apparent loss of amphetamine-stimulated substrate efflux, supporting a major role for this residue in the transport kinetic mechanism.

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          Most cited references72

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          SLC6 neurotransmitter transporters: structure, function, and regulation.

          The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na(+)/Cl(-)-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.
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            The prolyl isomerase PIN1: a pivotal new twist in phosphorylation signalling and disease.

            Protein phosphorylation regulates many cellular processes by causing changes in protein conformation. The prolyl isomerase PIN1 has been identified as a regulator of phosphorylation signalling that catalyses the conversion of specific phosphorylated motifs between the two completely distinct conformations in a subset of proteins. PIN1 regulates diverse cellular processes, including growth-signal responses, cell-cycle progression, cellular stress responses, neuronal function and immune responses. In line with the diverse physiological roles of PIN1, it has also been linked to several diseases that include cancer, Alzheimer's disease and asthma, and thus it might represent a novel therapeutic target.
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              How addictive drugs disrupt presynaptic dopamine neurotransmission.

              The fundamental principle that unites addictive drugs appears to be that each enhances synaptic dopamine by means that dissociate it from normal behavioral control, so that they act to reinforce their own acquisition. This occurs via the modulation of synaptic mechanisms that can be involved in learning, including enhanced excitation or disinhibition of dopamine neuron activity, blockade of dopamine reuptake, and altering the state of the presynaptic terminal to enhance evoked over basal transmission. Amphetamines offer an exception to such modulation in that they combine multiple effects to produce nonexocytic stimulation-independent release of neurotransmitter via reverse transport independent from normal presynaptic function. Questions about the molecular actions of addictive drugs, prominently including the actions of alcohol and solvents, remain unresolved, but their ability to co-opt normal presynaptic functions helps to explain why treatment for addiction has been challenging. Copyright © 2011 Elsevier Inc. All rights reserved.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                24 August 2012
                21 June 2012
                21 June 2012
                : 287
                : 35
                : 29702-29712
                Affiliations
                From the []Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202-9037 and
                the [§ ]Center of Physiology and Pharmacology, Institute of Pharmacology, Medical University Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria
                Author notes
                [3 ] To whom correspondence may be addressed. Tel.: 43-1-4277-64123; Fax: 43-1-4277-9641; E-mail: harald.sitte@ 123456meduniwien.ac.at .
                [4 ] To whom correspondence may be addressed. Tel.: 701-777-3419; Fax: 701-777-2382; E-mail: roxanne.vaughan@ 123456med.und.edu .
                [1]

                Both authors contributed equally to this work.

                [2]

                Both authors contributed equally to this work.

                Article
                M112.367706
                10.1074/jbc.M112.367706
                3436161
                22722938
                1d515f01-b0e9-4eb0-b346-e5d84a38c3b5
                © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Non-Commercial License applies to Author Choice Articles

                History
                : 29 March 2012
                : 13 June 2012
                Funding
                Funded by: National Institutes of Health
                Award ID: R01 DA13147
                Categories
                Neurobiology

                Biochemistry
                phospho-specific antibody,map kinases (mapks),proline-directed phosphorylation,sh3 domains,protein kinase c (pkc),mass spectrometry (ms),cis-trans isomerization,1-methyl-4-phenylpyridinium (mpp+),erk,pp1/2a

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